HSP20 Inhibits Amyloidogenesis and Neurotoxicity

ABSTRACT

The present invention compositions and methods of using at least a portions of an isolated and purified α-crystallin polypeptide that includes one or more β-pleated sheets and that prevents neurotoxicity and amyloidogenesis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a is a divisional patent application of U.S.Ser. No. 11/139,770 filed on May 26, 2005, which claims priority to U.S.provisional patent application 60/575,758 filed on May 26, 2004 andentitled “HSP20 Inhibits Amyloidogenesis and Neurotoxicity,” all ofwhich are hereby incorporated by reference in their entirety.

STATEMENT OF FEDERALLY FUNDED RESEARCH

The U.S. Government may own certain rights in this invention pursuant tothe terms of the NSF Grant No. BES-9734496.

TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to the field ofamyloidogenesis, and more particularly, to a heat shock protein withβ-pleated sheets that inhibits amyloidogenesis and neurotoxicity.

REFERENCE TO A SEQUENCE LISTING

The present application includes a Sequence Listing filed separately asrequired by 37 CFR 1.821-1.825.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is describedin connection with amyloidogenesis and neurotoxicity.

A number of pathophysiologic diseases have been classified as involving“amyloidosis,” which are characterized by the deposition of abnormalfibrils (“amyloid fibrils”) in extracellular spaces. The amyloid fibrilformation is thought to represent a final common result from themisfolding of a diverse array of proteins. Regardless of theirbiochemical composition, all types of amyloid fibrils share certaincharacteristics, including: a β-pleated sheet structure; greenbirefringence under polarized light after staining with Congo Red dye;and a fibrillar morphology in electron micrographs.

The deposition of amyloid fibrils affects several organs. One type ofamyloid fibril causes “cerebral amyloidosis,” which covers the Alzheimergroup of diseases, namely, Alzheimer's disease (e.g., pre-seniledementia, senile dementia); Alzheimer's disease associated with Down'ssyndrome; Alzheimer's disease associated with othercentral-nervous-system diseases, such as Parkinson's disorder; andcongophilic angiopathy (whether or not associated with Alzheimer'sdisease).

Presently, there is no effective therapy for cerebral amyloidosis, whichwas a fatal outcome following the onset of amyloid deposits. Alzheimer'sdisease (AD) is estimated to be the fourth or fifth leading cause ofdeath among North Americans. Accumulating biochemical, histological, andgenetic evidence supports the hypothesis that the 4 kDa β-amyloidprotein (Aβ) is an essential component in the pathogenesis ofAlzheimer's disease. Selkoe D J, Science 275:630-631 (1997); Hardy J,Proc Natl Acad Sci USA 94:2095-2097 (1997). Despite the intense interestin the role of Aβ in the etiology of AD, the role of Aβ in fibrilformation is poorly understood.

SUMMARY OF THE INVENTION

The present inventors have recognized and discovered that Hsp20, a novelα-crystallin isolated from the bovine erythrocyte parasite Babesiabovis, is able to prevent aggregation of amyloidogenic proteins. It wasfound that the α-crystallin isolated and purified from the bovineerythrocyte parasite Babesia bovis was able to prevent amyloidogenictarget proteins, e.g., β-Amyloid (Aβ), from aggregating when the Hsp20and the target protein were at or near equimolar levels. It was furtherfound that Hsp20 prevented Aβ amyloid formation at a wide range of molarratios of Hsp20 to Aβ, e.g., of about 1:1,000 to 1:200,000.Surprisingly, at higher concentrations of Hsp20, the protein no longerdisplays its aggregation inhibition and toxicity attenuation properties.The development of novel aggregation inhibitors is useful for thetreatment of neurodegenerative diseases and disorders, e.g.,Alzheimer's, Huntington's, and Parkinson's disease and prion proteins,that involve amyloid toxicity.

More particularly, the present invention includes an isolated andpurified polypeptide that includes a portion of an α-crystallinpolypeptide having one or more β-pleated sheets that inhibitsneurotoxicity and amyloidogenesis. For example, the polypeptide may benon-human, such as a polypeptide from a bovine erythrocyte parasite. Oneparticular example of a used to prevent neurotoxicity andamyloidogenesis may be at least a portion of an Hsp20 polypeptide from aBabesia sp. bovine erythrocyte parasite, e.g., Babesia bovis. In oneexample, the present invention includes both the nucleic acid (SEQ IDNO.:1) and/or the amino acid sequences of the Hsp20 protein (SEQ ID NO.:2), truncations and fusion proteins thereof. For example, the Hsp20polypeptide may include a tag that permits rapid isolation, e.g., aHis-tag and even a protease cleavage site, as are well-known to skilledartisans.

The present invention also includes a method of making an α-crystallinpolypeptide by culturing a host cell into which a nucleic acid sequenceencoding at least a portion of the polypeptide of SEQ ID NO.: 2 has beenintroduced in transient, selective or permanent form under conditionswherein the host cell expresses the polypeptide and wherein thepolypeptide exhibits α-crystallin activity, attenuated neurotoxicity andinhibits amyloidogenesis. An isolated and purified polypeptide may beexpressed and isolated by the methods disclosed herein.

One example of a protein for use with the present invention is anisolated and purified, small heat shock protein that includes one ormore β-pleated sheets that modulates neurotoxicity over about aone-thousand-fold concentration range by stabilizing an amyloidogenicprotein. The concentration range for the activity of the reduction inneurotoxicity of target protein is between about 1 nM to about 5 μM. Inanother example, the molar ratio of heat shock protein to amyloidogenicprotein may be between about 0.0005 to about 0.001. These proteins maybe used in a method of stabilizing a protein with one or more β-pleatedsheet by contacting the protein with a heat shock polypeptide with oneor more β-pleated sheets wherein the protein remains stable and exhibitsattenuated neurotoxicity over a one-thousand-fold concentration range.

The present invention may also be used in a method for stabilizing aprotein with a β-pleated sheet in which the target protein is contactedwith a heat shock polypeptide that includes one or more β-pleated sheetswherein the protein remains stable over a one-thousand-foldconcentration range at a high temperature. The heat shock polypeptidemay be an Hsp20 that exhibits α-crystallin activity and inhibits theformation of Aβ multimers such as a parasitic, bacterial, viral, fungalor mammalian analog of Hsp20 that exhibits α-crystallin activity andattenuated toxicity to Aβ. The small, heat shock protein (andtruncations thereof), may be used in a method of reducing proteinaggregation by contacting an aggregation of proteins with an Hsp20wherein the protein aggregation is reduced. The proteins of the presentinvention, either with or without a tag, may be used in, e.g., a methodof treating a medical condition that exhibits protein aggregation byadministering to a subject a physiological effective amount of a heatshock protein with one or more β-pleated sheets, whereby the proteinreduces protein aggregation levels in a neuronal tissue of the subject.

The method of preventing protein aggregation disclosed herein includescontacting an amyloidogenic protein with an effective amount of at leasta portion of the polypeptide of SEQ ID NO.: 2. The amyloidogenic proteinmay be in, at or about a tissue, e.g., in, at or about a neural cell.The protein for preventing aggregation includes an isolated and purifiedportion of a polypeptide having a significant similarity to theα-crystallin family of proteins and one or more β-pleated sheetsisolated from Babesia sp., e.g., full-length Hsp20, or amino, carboxy,or amino and carboxy terminus truncated Hsp20 protein that inhibits,e.g., Aβ₁₋₄₀ fibril formation.

Yet another embodiment of the present invention is a kit or dosage formthat includes a first container having an isolated or purifiedpolypeptide with α-crystallin activity and one or more β-pleated sheetsthat inhibits neurotoxicity and amyloidogenesis. For example, apharmaceutical composition for treating amyloidogenesis may include aneffective amount of at least a portion of a purified polypeptide withα-crystallin activity and one or more β-pleated sheets that inhibitsneurotoxicity and amyloidogenesis in pharmaceutically acceptablecarrier. One such isolated and purified polypeptide includes anα-crystallin, small, heat shock protein Hsp20 having one or moreβ-pleated sheets that inhibits neurotoxicity and amyloidogenesis, e.g.,one isolated and purified (from the organism or as a recombinantproduct) from a Babesia bovis. The polypeptide may also include a tagfor isolation, e.g., an MBP, myc, GST, His-tag and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIG. 1 is a graph of a turbidity assay used to quantitate the ability ofHsp20 to solubilize target proteins at elevated temperatures;

FIG. 2 is a graph that shows percent reduction of light scattering ofvarious solutions of ADH and Hsp20 relative to ADH alone;

FIG. 3 is a graph of representative Congo Red absorbance spectra;

FIG. 4 is a graph that shows the effect of Hsp20 on reduction of Aβfibril formation at different concentrations of Aβ and Hsp20;

FIGS. 5A to 5C are electron micrographs of Aβ fibrils, Hsp20, andAβ-Hsp20 mixtures, respectively;

FIG. 6 is a graph that plots cell viability as a function of Hsp20concentration;

FIG. 7 is a graph of turbidity of protein aggregates as a function oftime;

FIG. 8 is a nucleic acid sequence of an Hsp20 for use with the presentinvention (SEQ ID NO.: 1);

FIG. 9 is the amino acid sequence of an Hsp20 (SEQ ID NO.: 2) for usewith the present invention in accordance with SEQ ID NO.: 1;

FIG. 10 is a secondary structure prediction of Hsp20;

FIG. 11 is a graph of CD measurements carried out on an AVIV model 62;

FIGS. 12A to 12C show the activities of small heat shock proteins(sHsps) to prevent Aβ aggregation and toxicity. All protein samplescontaining Aβ and sHsps were mixed for 24 hours before aggregation andtoxicity assay. Congo red was used as an indicator of Aβ aggregation andFACS array was used for the biological activities using dye. (12A) 100μM Aβ with His-Hsp20 (solid triangles: Congo red assay, blank triangles:biological activities); (12B) 20 μM Aβ with His-Hsp17.7 (solid circles:Congo red assay, blank circles: biological activities); and (12C) 100 μMAβ with Hsp27 (solid squares—Congo red assay, blank squares—biologicalactivities);

FIG. 13 and FIG. 14 show the kinetics of Aβ aggregation with sHsps. Aβwas incubated with or without sHsps and the aggregation rates weredetected by FIG. 13 Congo red, and FIG. 14 Turbidity at 405 nm (darkgray diamonds—100 μM Aβ, black triangles—100 μM Aβ and 0.1 μM His-Hsp20,gray circles—100 μM Aβ and 0.1 μM His-Hsp17.7, light gray squares—100 μMAβ and 0.1 μM Hsp27);

FIG. 15 and FIG. 16 show representative electron micrograph images of Aβwith sHsp. The samples were mixed for 2 hours and then the pictures weretaken; (FIG. 15) 100 μM Aβ and 0.1 μM His-Hsp20; (FIG. 16) 100 μM Aβ and0.1 μM His-Hsp17.7, the length of scale bar is 100 nm;

FIG. 17 is a graph that shows the kinetics of fibril formation in thepresence and absence of hsp20 Fibril formation as a function of time inthe presence and absence of Hsp20 (100 μM Aβ and 100 nM Hsp20 were used;solutions were incubated with mixing at 37° C. and fibril formation wasdetermined using Congo red binding);

FIG. 18 shows the dynamic relationship of the oligomeric states of hsp20using FPLC analysis of 29 μM hsp20 in 500 mM NaCl (13.69=16 mer;15.05=10 mer; 16.85=hexamer; 17.99=tetramer; 20.18=dimer; 22.72=monomerDimer=15% of total);

FIG. 19 is a graph that shows the dynamic relationship of the oligomericstates of hsp20;

FIG. 20 is a sequence alignment of M.j-sHSP and B. bovis HSP20: Nterminal and C terminal deletions with the C-terminal deletion ofHSP20=EMRRVQIDAKA (SEQ ID NO.: 5);

FIG. 21 is a graph of the size distribution of truncated Hsp20 (tHsp20);

FIG. 22 shows that truncated hsp20 blocks ADH denaturation;

FIG. 23 is a graph that shows that fibril formation of Aβ peptide in thepresence of full length and truncated hsp20.

FIG. 24 includes the amino acid sequences for the full length, aminoterminal truncated, carboxy terminal truncated, both the amino andcarboxy terminal truncated, a his-tagged full length, a his-tagged aminoterminal truncated, a his-tagged carboxy terminal truncated, and ahis-tagged the amino and carboxy terminal truncated protein;

FIG. 25 is a gel that shows the effect of the truncations (as listed) onthe target protein, and that the monomeric protein is depleted over timeindicating its role in multimer formation; and

FIG. 26 is a gel that shows the effect of the truncations (as listed) onthe target protein, and that the monomeric protein is depleted over timeindicating its role in multimer formation.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

β-Amyloid (Aβ) is a major protein component of senile plaques inAlzheimer's disease, and is neurotoxic when aggregated. The size ofaggregated Aβ responsible for the observed neurotoxicity and themechanism of aggregation are still under investigation; however,prevention of Aβ aggregation still holds promise as a means to reduce Aβneurotoxicity. Alzheimer's disease (AD), the leading cause of dementiain the aging population, is accompanied by the accumulation of β-amyloidpeptide (Aβ) as amyloid fibrils in senile plaques in the cerebralcortex. It is suggested by many that Aβ contributes to theneurodegeneration associated with AD via the toxicity of the peptide inaggregated form.

The superfamily of small heat shock proteins, sHSPs, are a diverse groupof proteins involved in prevention of protein misfolding and the onsetof programmed cell death (16, 17). They typically exist as largemultimeric complexes containing 4 to 40 subunits, with molecular massesof 40 kDa or less per subunit. The sHsps bind to their substrates withhigh affinity and prevent protein misfolding in an ATP independentreaction (18). Generally, the α-crystallins are considered to be sHsps(15, 19). Circular Dichroism spectroscopy (CD) studies predict thatβ-pleated sheet dominates the secondary structure of the α-crystallins(20, 21). A conserved 90 amino acid sequence, located near theC-terminus, termed the α-crystallin domain, is the hallmark of allα-crystallins.

Other small heat shock proteins and α-crystallins have been examined fortheir ability to prevent Aβ and other amyloid fibril formation, withgood success at relatively low molar ratios of α-crystallin to amyloid(11, 12, 22, 23). However, in none of these studies has toxicityinhibition been reported. Indeed, in work reported by others (12),αB-crystallin prevented Aβ fibril formation but enhanced toxicity. TheHsp20 protein reported herein, both reduces Aβ fibril formation at verylow molar ratios, and reduces Aβ toxicity at similar molar ratios.

The present inventors have recognized and discovered that Hsp20, a novelα-crystallin isolated from the bovine erythrocyte parasite Babesia sp.,e.g., Babesia bovis, is able to prevent aggregation of amyloidogenicproteins. Other members of the Babesia family, e.g., Babesia bigemina,also express Hsp20 with very high amino acid sequence conservation. Itwas found that the α-crystallin isolated and purified from the bovineerythrocyte parasite Babesia bovis prevented denaturation of alcoholdehydrogenase when the two proteins are present at near equimolarlevels. The same assay may be used to initially screen other species.Next, the ability of Hsp20 to prevent Aβ amyloid formation wasinvestigated using Congo Red binding. It was found as demonstratedherein that not only is Hsp20 able to dramatically reduce Congo Redbinding, but it was also able to do so at molar ratios of Hsp20 to Aβ of1 to 1000. Electron microscopy confirmed that Hsp20 does prevent Aβfibril formation. Hsp20 was also able to significantly reduce Aβtoxicity to both SH-SY5Y and PC12 neuronal cells at similar molarratios. Surprisingly, at higher concentrations of Hsp20, the protein nolonger displays its aggregation inhibition and toxicity attenuationproperties. The development of novel aggregation inhibitors is usefulfor the treatment of neurodegenerative diseases and disorders, e.g.,Alzheimer's disease, that involve amyloid toxicity.

Investigation of the relationship between aggregated Aβ peptide size,structure and toxicity is ongoing. In an aggregated state containingfibrils, protofibrils, and low molecular weight intermediates/oligomers,Aβ peptide has proven to be toxic to cultured neuronal cells (1, 2). Aβstructures reported to be toxic include a non-fibrillar species ofapproximately 17 to 42 kDa (1), protofibrils species with hydrodynamicradii on the order of 6 to 9 nm β) and 22-35 nm or 97-367 nm (4) andfibril species, with some investigators suggesting that the smalleroligomeric Aβ species are more toxic than fibril and protofibril forms(5). Many believe that one strategy for preventing neurodegenerationassociated with AD is the prevention of aggregation of Aβ into its toxicoligomeric or fibril forms.

Inhibitors of Aβ aggregation have been investigated with the aim ofpreventing Aβ toxicity (6-9). Some of these inhibitors are smallpeptides with sequences that mimic the sequence of Aβ believed to beessential for aggregation and fibril formation (8). Peptide inhibitorsof this class have been able to prevent Aβ toxicity by altering theaggregated structure when added at inhibitor to Aβ molar ratios of 1:1(8, 10). The use of molecular chaperones including α-crystallins andother small heat shock proteins has also been explored as a means ofpreventing Aβ aggregation and toxicity. For example, human sHsp27inhibited Aβ1-42 fibril formation (11). When αB crystallin was examined,it was actually found to increase toxicity and 13 pleated sheet contentof Aβ1-40 although it prevented fibril formation of Aβ in vitro atinhibitor to Aβ molar ratios of 1:1 or 1:5 (12).

The present invention is a novel small heat shock protein, Hsp20,isolated from the erythrocyte parasite Babesia bovis to preventaggregation of Aβ and Aβ toxicity. It is shown herein that Hsp20 hasα-crystallin-like properties, and that it prevents aggregation ofdenatured alcohol dehydrogenase. Hsp20 also prevents amyloid formationof Aβ as indicated by Congo Red binding at molar ratios of Hsp20 to Aβof 1:1000. Hsp20 attenuated the toxicity of Aβ in SH-SY5Y and PC12neuronal cells at analogous molar ratios. Furthermore, Hsp20 appears tobe able to prevent Aβ aggregation via a novel mechanism and at muchlower concentrations than what has been necessary to prevent aggregationwith other inhibitors. Hsp20 may be a useful molecular model for thedesign of the next generation of Aβ aggregation inhibitors to be used inthe treatment of AD.

Materials. Aβ(1-40) was purchased from Biosource International(Camarillo, Calif.). Cell culture reagents were purchased from GibcoBRL(Grand Island, N.Y.). All other chemicals were obtained from Sigma (St.Louis, Mo.) unless otherwise stated. His-tagged-Hsp20 was produced andisolated in E. coli. Previously, Hsp20 had been made as a fusion proteinwith Maltose binding protein (MBP), however, it was found that theMBP-Hsp20 did not show the effects described and claimed herein (13).

Aβ Peptide Preparation. Stock solutions of 10 mg/ml Aβ(1-40) peptideswere dissolved in 0.1% (v/v) trifluoroacetic acid (TFA) in water. Afterincubation for 2030 min. at 25° C., the peptide stock solutions werediluted to concentrations of 0.5 mg/ml by addition of sterile phosphatebuffered saline (PBS; 0.01 M NaH₂PO₄, 0.15 M NaCl, pH 7.4). The peptideswere diluted to final concentrations of 20 μM, 50 μM, and 100 μM byaddition of PBS for Congo Red and electron microscopy (EM) studies. Cellculture medium (below) replaced PBS in toxicity studies. These solutionswere rotated at 60 revolutions per minute at 25° C. for 24 hours toensure aggregation.

Cell Culture. Human neuroblastoma SH-SY5Y cells (obtained from Dr.Evelyn Tiffany-Castiglioni, College of Veterinary Medicine, Texas A&MUniversity, College Station, Texas) were cultured in minimum essentialmedium (MEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 3 mML-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2.5 μg/mlamphotericin B (fungizone) in a humidified 5% (v/v) CO₂/air environmentat 37° C. Rat pheochromocytoma PC12 cells (ATCC, Rockville, Md.) werecultured in RPMI medium supplemented with 10% (v/v) horse serum, 5%(v/v) FBS, 3 mM L-glutamine, 100 U/ml penicillin, 100 μg/mlstreptomycin, and 2.5 μg/ml fungizone in a humidified 5% (v/v) CO₂/airenvironment at 37° C. For the toxicity assays, cells were plated at adensity of 1×10⁵ cells/well in 96 well plates and aggregated peptidesolutions were added to the cells 24 hours after plating.

Congo Red Binding. Congo Red studies were performed to assess thepresence of amyloid fibrils in Aβ solution. Congo Red dye was dissolvedin PBS to a final concentration of 120 μM. Congo Red solution was addedto the peptide solutions at the ratio of 1:9. The peptide solution andcontrol solution were allowed to interact with Congo Red for 30˜40minutes prior to absorbance measurement with a Model 420 UV-Visspectrophotometer (Spectral Instruments, Tucson, Ariz.) at 25° C. Thefibril formation of the samples was estimated from the absorbance usingequation (1);

[Aβ _(Fib)]=(⁵⁴¹ A _(t)/4780)−(⁴⁰³ A _(t)/6830)−(⁴⁰³ A _(CR)/8620)  (1)

where [Aβ_(Fib)] is the concentration of Aβ fibril, ⁵⁴¹A_(t), ⁴⁰³A_(t)and ⁴⁰³A_(CR) are the absorbances of the sample and Congo Red at thewavelength of 541 nm and 403 nm, respectively (14). From these data,relative fibril concentrations were calculated as the ratio of samplefibril concentration to pure Aβ fibril concentration.

MTT Reduction Assay. Cell viability was measured 24 hours after peptideaddition to cells using the 3, (4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) reduction assay. 10 μl of 12 mMMTT was added to 100 μl of cells plus medium in a 96 well plate. Thecells were incubated with MTT for 4 hours in a CO₂ incubator. Then, 100μl of a 5:2:3 N,N-dimethylforamide (DMF): sodium dodecyl sulfate (SDS):water solution (pH 4.7) was added to dissolve the formed formazancrystals. After 18 hours of incubation in a humidified CO₂ incubator,the results were recorded by using an Emax Microplate reader at 585 nm(Molecular Devices, Sunnyvale, Calif.). Percentage cell viability wascalculated by comparison between the absorbance of the control cells andthat of peptide or peptide:Hsp20 treated cells. Relative cell viabilityincrease, was calculated from the ratio of the difference in cellviability of Hsp20:Aβ treated cells and cells treated with pure Aβ,divided by viability of cells treated with pure Aβ.

Electron Microscopy (EM). 200 μl of Aβ peptide solution, prepared asdescribed above, was mixed, placed on glow discharged grids, and thennegatively stained with 1% aqueous ammonium molybdate (pH 7.0). Gridswere examined in a Zeiss 10 C transmission electron microscope at anaccelerating voltage of 80 kV. Calibration of magnification was donewith a 2,160 lines/mm crossed line grating replica (Electron MicroscopySciences, Fort Washington, Pa.).

Turbidity Assay. ADH Turbidity Assay. Light Scattering of ADH and Hsp20was performed as previously described (15). Briefly, the aggregation ofADH and Hsp20 in solution was measured by the apparent absorption due toscattering at 360 nm in a Gilford Response II spectrophotometer at 58°C. Solutions were mixed at room temperature in 50 mM phosphate buffer,pH 7.0 and analyzed immediately. 1.625 μM ADH was used in all studies.Hsp20 concentrations were varied to obtain different molar ratios ofHsp20 to ADH. Absorbance readings were taken at 1 minute intervals forone hour.

Aβ Turbidity Assay. Aβ samples were prepared as described for otherstudies, however, aggregation took place without mixing at 4° C. Theseconditions were chosen to slow down the rate of aggregation. Hsp20 wasadded to Aβ samples either at the beginning of incubation or after 6days (a time insufficient for large changes in turbidity to beobserved). Turbidity was determined by measuring sample absorbance at400 nm on the UV-vis spectrophotometer.

Statistical analysis. For each study, at least 3 independentdeterminations were made. Significance of results was determined via thestudent t test with p<0.05 unless otherwise indicated. Data are plottedas the mean plus or minus the standard error of the measurement.

Characterization of Hsp20. Hsp20 is a 177 amino acid, 20.1 kDa proteinisolated from Babesia sp., specifically Babesia bovis, a protozoanbovine erythrocyte parasite (13). Initial characterization by BLASTsearch of the NCBI database indicated sequence similarity with theα-crystallin family of small heat shock proteins. Hsp20 contains aregion near its C-terminus that corresponds to the ˜95 amino acidα-crystallin domain common to members of this protein family. Theseproteins are thought to be involved in the cellular response to stress.Previous work (Ficht, unpublished observation) has revealed that Hsp20expression is up-regulated in times of thermal, nutritional andoxidative stress to the organism. Based on the apparent involvement ofHsp20 in the stress response coupled with its homology to theα-crystallins, α-crystallin activity assays were applied to Hsp20.

For the His-Tag Hsp20 expression construct, the following primers wereused: Primer: F32901FT forward primer for hsp20 with Tobacco Etch Virus(TEV) protease site immediately upstream:

(SEQ ID NO.: 3) 5′-GAA AAT CTT TAT TTT CAA GGT ATG TCG TGT ATT ATG AGG TGC-3′;The first seven (7) codons encode the TEV protease site, the last seven(7) codons are the beginning of the hsp20 coding sequence:

Primer: F32901RT Reverse nrimer for hsp20 with in-frame stop:

(SEQ ID NO.: 4) 5′-CTA TTA GGC CTT GGC GTC AAT CTG AAC-3′;There primers were used to PCR a 621 coding region.

Ligation/Transformation of above insert. The Invitrogen protocol pTrcHisand pTrcHis2 TOPO TA Expression Kits Version I were used to ligate andtransform the PCR product, relevant portions and solutions incorporatedherein by reference. The PCR reactions were incubated directly with theTOPO plasmid according to the manufacture's protocol. This resulted inligation of the PCR product into the pTrcHis vector. Transformed vectorinto chemically competent TOP10 cells (Invitrogen) and screened onLB/amp50 plates.

Directional PCR of transformants to screen for possible clones. TheProBond Purification System protocol version I from Invitrogen was used.The Hybrid protocol was modified and used to purify Hsp 20 from thelysate. Cells were grown to OD600 of 0.5 and induced with 1 mM IPTG for5 hours, spun down and the removed media. The cells were lysed in PBSusing a French pressure cell, the lysate centrifuged and the supernatantremoved. The pellet containing hsp20 was suspended in Denaturing BindingBuffer (in accordance with manufacturer's instructions, relevantportions and solutions incorporated herein by reference) containing 8Murea.

Hybrid purification procedure. Briefly, resin was washed with buffercontaining 8M urea. Incubated resin with lysate pellet in 8M urea forone hour. The resin was washed with denaturing buffer and then washedwith native wash buffer (no urea). Next, the protein was eluted from theresin using 100 mM EDTA in water. Dialyzed over night into PBS pH 7.0.At this point, the protein can be stored at either 4 or 25 deg. C.Frozen samples are first diluted with glycerol (20% final).

FIG. 1 is a graph of a turbidity assay used to quantitate the ability ofHsp20 to solubilize target proteins at elevated temperatures. Turbiditywas measured using Light scattering of ADH, as determined by absorbanceat 360 nm, at elevated temperature in the presence and absence of Hsp20.ADH at a concentration of 1.625 μM is incubated in phosphate bufferalone (open circles) and in the presence of 5.75 μM Hsp20 (closedtriangles) at 58° C. for one hour.

A solution of alcohol dehydrogenase when incubated at 58° C. for 60minutes exhibits a dramatic increase in light scattering due todenaturation of the enzyme. When Hsp20 was included in the solution attime zero in a molar ration of 2:1 Hsp20:ADH, a 2.3 fold reduction inlight scattering is observed at 60 minutes. To determine the mosteffective molar ratio for the reduction of light scattering, a series ofstudies applying different molar ratios was conducted.

FIG. 2 is a graph that shows percent reduction of light scattering attime infinity of various solutions of ADH and Hsp20 relative to ADHalone. The greatest reduction in light scattering was observed at 2:1molar ratio (Hsp20:ADH). All studies were performed at 58° C. ADH wasincluded at 1.625 μM in each study. Light scattering at time infinitywas estimated from absorbance versus time curves for each mixture. Attime infinity (as determined by curve fitting software), the greatestreduction in light scattering is seen at a molar ratio of 2:1 (Hsp20 toADH). Having established the α-crystallin activity of Hsp20 basedstandard assays, the ability of Hsp20 to affect Aβ(1-40) fibrilformation was determined.

Effect of Hsp20 on Aβ Fibril Formation Prevention. Given that Hsp20 wasable to prevent aggregation of denatured ADH, its ability to preventaggregation and amyloid fibril formation of Aβ(1-40) was determined.Congo Red binding was used as an indicator of amyloid formation. Inthese studies μ50 μM μconcentrations of Aβ(1-40) were used to createfibrils. Hsp20 at concentrations from 50 μM to 2.5 μM were added toAβ(1-40) solutions prior to aggregation. This corresponds to molarratios of Hsp20 to Aβ of 1:1,000,000 to 1:20. After 24 hours ofincubation, sufficient time for typical fibril formation, Congo Red wasadded to samples to assess fibril formation.

FIG. 3 is a representative absorbance spectra of Congo Red with Aβ andAβ-Hsp20 mixtures. 100 μM Aβ(1-40) was used in all cases. Hsp20 wasadded at the beginning of the 24 hour aggregation at room temperaturewith mixing. Hsp20 alone did not alter Congo Red absorbance. PureAβ(1-40) solutions caused a characteristic shift in absorbance to longerwavelengths and an increase in intensity. Hsp20, when added to Aβ priorto aggregation, attenuated the shift in absorption and increase inintensity associated with Aβ fibril formation.

FIG. 4 is a graph that summarizes the effects of Hsp20 on reduction ofAβ fibril formation at different concentrations of Aβ and Hsp20. AtHsp20 concentrations below 0.41M, Aβ fibril formation decreased as afunction of Hsp20 concentration. However, Hsp20 became less effective atpreventing Aβ fibril formation in the higher concentration range. Theconcentration of Hsp20 needed for optimal prevention of fibril formationappeared to be a function of Aβ concentration, with the lowestconcentration of Aβ exhibiting the optimum in prevention of fibrilformation at the lowest Hsp20 concentration. Fibril formation isreported relative to fibril formation seen with pure Aβ solutions. Meanplus or minus standard deviation of at least 3 independent studies areshow (open circles—20 μM Aβ: squares—50 μM Aβ: triangles—100 μM Aβ).

The optimum in fibril formation for the concentration of Aβ testedoccurred at the molar ratios between 0.000005 and 0.001 of Hsp20 to Aβ.The molar ratio of Hsp20 to Aβ needed to optimally prevent fibrilformation is several orders of magnitude lower than that needed toprevent aggregation of ADH.

FIGS. 5A to 5C are electron micrographs of Aβ fibrils, Hsp20, andAβ-Hsp20 mixtures that correspond to conditions used in the Congo Redbinding studies. These representative electron micrographs of Aβ andHsp20 were taken after 24 hours of aggregation (5A) 100 μM Aβ, (5B) 0.1μM Hsp20, and (5C) 100 μM Aβ+0.1 μM Hsp20. In the images, 100 μM Aβ and0.1 μM Hsp20 were used. As seen in FIG. 5A, Aβ formed long individualfibrils and groups of long fibrils under the aggregation conditionsemployed. Hsp20, at 0.1 μM and room temperature, formed species ofapproximately 13.6 nm in diameter, composed of 2 subunits, 5.4 nm inwidth each, with a distance of 2.7 nm between subunits (FIG. 5B). Inmicrographs of the Aβ-Hsp20 mixture (FIG. 5C), small globular specieswith a 16.3 nm diameter and variable length were observed. These speciescould be an Hsp20-Aβ complex, large Hsp20 aggregates, and/orAβ-protofibrils. Aβ fibrils were noticeably absent from micrographs ofAβ-Hsp20 mixtures.

Ability of Hsp20 To Prevent Toxicity of Aβ. Aβ is typically toxic toneuron-like cells (SY5Y cells, and PC12 cells etc.) when in aggregatedform. The effect of Hsp20 on Aβ toxicity was measured using the MTTassay for 100 μM Aβ added to SH-SY5Y cells and 2 μM Aβ added to PC12cells.

FIG. 6 is a graph that plots cell viability as a function of Hsp20concentration. In FIG. 6, the relative cell viability of SY5Y cells(triangles) and PC12 cells (circles) treated with 100 μM (triangles) and2 μM (circles) Aβ as a function of Hsp20 concentration is shown.Viability was measured via the MTT reduction assay. N is greater than orequal to 6. Aβ(1-40) was incubated for 24 hrs. in the media prior toaddition to the cells. Viability as a function of molar ratio of Hsp20to Aβ; viability is reported relative to Aβ treated cells. Hsp20 byitself had no effect on SH-SY5Y or PC12 cell viability. However, Hsp20,when added to Aβ prior to aggregation, had a profound effect on Aβtoxicity observed in both cell types. It appears that the two viabilitycurves as a function of Hsp20 to Aβ molar ratio are superimposed. Themolar ratio of Hsp20 to Aβ needed for optimal toxicity attenuation isapproximately the same as the molar ratio needed for optimal fibrilformation prevention.

Ability of Hsp20 to reverse Aβ aggregation. In studies parallel to thosedescribed above, the ability of Hsp20 to reverse fibril formation, onceAβ was aggregated, was determined. Under conditions analogous to thosedescribed previously, and after 24 hours of Aβ aggregation, Hsp20 wasadded at optimal concentrations to prevent fibril formation (100 nM),incubated for up to 2 days, and then assessed fibril content via CongoRed binding. At all time points measured, fibril formation afteraddition of Hsp20 was equal to or greater than fibril formation of Aβalone after 24 hours aggregation (data not shown). In all cases,viability of SH-SY5Y cells, as measured by the MTT assay, treated withsolutions in which Hsp20 was added after 24 hour Aβ aggregation was notsignificantly greater than viability of cells treated with Aβ that hadbeen allowed to aggregate for 24 hours (data not shown).

Ability of Hsp20 to prevent later steps in Aβ aggregation. In order toassess if Hsp20 could prevent Aβ aggregation, even if added after thebeginning of aggregation, a second set of aggregation conditions wasdeveloped in which initial aggregation events (such as fibrilnucleation) may occur more slowly. 100 μM Aβ were allowed to aggregateat 4° C. without mixing and measured aggregation via changes inturbidity of the Aβ solution.

FIG. 7 is a graph of turbidity of protein aggregates as a function oftime. Aβ was allowed to aggregate under these conditions, a rapid risein turbidity was observed after 6 days incubation. Samples are incubatedat 4° C. without rotation (closed circles—100 μM Aβ(no Hsp20): opencircles). Hsp20 was added to 100 μM Aβ at the beginning of Aβincubation: triangles—Hsp20 addition to 100 μM Aβ at 6 day after Aβincubation.

When Hsp20 was added (final concentration of 100 nM) at the beginning ofaggregation, no dramatic increase in turbidity was observed. Theseresults are consistent with Congo Red and electron microscopy resultsobtained under rapid aggregation conditions. Similarly, when Hsp20(final concentration of 100 nM) was added to the Aβ solutions at day 6,again no dramatic increase in turbidity was observed.

Electron micrographs of samples prepared under slow Aβ aggregationconditions clearly indicated the presence of fibrils both in thepresence and absence of Hsp20, however, possibly less lateral fibrilaggregation was observed in solutions containing Hsp20 (micrographs notshown).

Toxicity of Aβ solutions prepared via slow aggregation (at 4° C. withoutmixing) with and without Hsp20, was measured. In all cases, toxicity waslargely unchanged upon addition of Hsp20, regardless of when Hsp20 wasadded in the aggregation process (data not shown).

The Hsp20 isolated, purified and characterized herein from the bovineerythrocyte parasite Babesia bovis (13) was demonstrated to haveα-crystallin activity in vitro. The apparent light scattering of alcoholdehydrogenase in solution at 58° C. is reduced when in the presence ofequimolar concentrations of recombinant Hsp20 (FIG. 1). This indicatesreduced aggregation of denatured ADH at the elevated temperature due toan interaction with Hsp20. The optimal Hsp20 to ADH binding ratio is 2:1(FIG. 2), consistent with the stoichiometry of binding one Hsp20 dimerto one ADH molecule. This is consistent with the observations of vanMontfort et al (20) that other alpha crystallins are active as dimers.In addition, Hsp20 is upregulated under heat shock conditions (42° C.)and oxidative stress (molecular O₂ and H₂O₂); and, as otherα-crystallins, it also forms higher order complexes as revealed throughdynamic light scattering analysis (unpublished observation). Hsp20 haslimited nucleotide and amino acid sequence homology to members of theα-crystallin family, with the majority of the identical/conserved aminoacids occurring in the region corresponding to the α-crystallin domain(13). The α-crystallin-like activity and ability to prevent proteinaggregation of Hsp20 indicate that it may be potentially useful as anaggregation inhibitor (or a model for an aggregation inhibitor) for Aβ.In view of its divergent sequence, this α-crystallin may have entirelyunique properties.

As show herein in FIGS. 3-5A-5C, that Hsp20 prevents Aβ fibril formationas indicated by the absence of Congo Red binding and of visible fibrilsin electron micrographs, at molar ratios near 1:1000 Hsp20 to Aβ. Thisis in sharp contrast to the near 1:1(2:1, or even 4:1 if ADH isconsidered a dimer) molar ratio needed to prevent ADH aggregation. Anumber of investigators have explored the ability of other small heatshock proteins, molecular chaperones, and α-crystallins to preventfibril formation in several systems including Aβ (11, 12, 22, 23). Mosthave found that the chaperones or small heat shock proteins can inhibitAβ aggregation at molar ratios of chaperone to Aβ of 1:10 to 1:100. Thelow concentrations of Hsp20 and other α-crystallins needed to preventfibril formation, relative to that needed to prevent ADH aggregation,could be related to specificity of the α-crystallins for β-sheet orfibril forming proteins. Alternatively, the difference in molar ratiosneeded to prevent ADH aggregation relative to those needed to prevent Aβfibril formation may be related to the temperature differences at whichthe studies were carried out. The ADH aggregation studies were carriedout at elevated temperature, while the Aβ fibril formation studies werecarried out at room temperature. The quaternary structure ofα-crystallins are generally temperature dependent; however, most often,more highly active oligomers (dimers) are formed at higher temperatures(20, 24).

Hsp20 may interact with an oligomer of Aβ in a near 1 to 1 molar ratio;however, the Aβ oligomer may be very dilute in Aβ solutions. In modelsof Aβ aggregation, investigators postulate that Aβ forms micelles ormultimeric nuclei from Aβ monomers or dimers via a high order process(25-27). It is from these micelles or nuclei that fibril growth isinitiated. Micelles or nuclei would be far less abundant than monomericAβ. Hsp20 binding may lead to removal of fibril initiating species fromsolution, thereby preventing Aβ fibril formation.

As seen in FIG. 4, there is an optimum concentration of Hsp20 for Aβfibril formation prevention. Oligomerization dynamics of α-crystallinsare concentration dependent (28). Hsp20 may aggregate to form a lessactive structure at higher concentrations. High molecular weightoligomers of heat shock proteins, which tend to form at highconcentrations, are less active than the low molecular weight oligomers(29). Additionally, equilibrium distribution of oligomeric Hsp20 speciesare likely to dependent upon conformers or oligomers of substrate Aβpresent in solution.

Analogous to fibril formation prevention data, Hsp20 attenuates Aβtoxicity at similar molar ratios (FIG. 6). These data suggest that theAβ species which bind Congo Red and have fibril appearance on electronmicrographs are toxic. Non-fibrillar ADDLs, low molecular weightoligomers, protofibrils, and fibrils have all been found to be toxic (1,3). In some cases oligomeric Aβ has been found to be more toxic thanfibril Aβ (5). These results suggest that addition of Hsp20 leads to areduction in the formation of some of these species.

A careful examination of electron micrographs of Aβ-Hsp20 mixtures underoptimal fibril formation and toxicity prevention conditions (FIG. 5A-5C)reveals the presence of small globular species with a 16.3 nm diameterand variable length. Size analysis of these globular structures suggeststhat the species may be an Hsp20-Aβ complex rather than Aβ protofibrilssince the width of these species (16.3 nm) is atypical for Aβ micelles(5˜11 nm), protofibrils (4˜8 nm), or fibrils (6˜10 nm) (27).

When Aβ was aggregated slowly at 4° C., Hsp20 was able to prevent largeincreases in turbidity when added to Aβ at a 1:1000 molar ratio of Hsp20to Aβ (FIG. 7). Electron micrographs of species formed indicate thatunder these conditions Hsp20 prevented some lateral aggregation offibrils but did not prevent fibril formation. In addition, with slowaggregation, Hsp20 did not prevent toxicity. Thus, lateral aggregationof fibrils or its prevention does not appear to be associated withtoxicity. These results are consistent with reports by others that Aβoligomers are more toxic that Aβ fibrils, and that a variety ofsub-fibril sized species are toxic (1-5). The inability of Hsp20 toprevent Aβ fibril formation under the conditions used may be due totemperature affects on Hsp20 oligomerization. At 4° C., there may beinsufficient active Hsp20 dimers present to prevent fibril formation orhsp20 may be subject to cold denaturation and inactivation at 4° C.(data not shown). Alternatively, the energetics of the Hsp20-Aβinteraction may be adversely affected by the drop in temperature. Inaddition, under slow aggregation conditions, the relative abundance ofthe Aβ species that bind to Hsp20 may be significantly different thanunder rapid aggregation conditions under which the Hsp20 optimumconcentration was determined.

A number of different approaches have been explored for prevention of Aβamyloid fibril formation and toxicity. Pentapeptides such as SEQ ID NO.:6 KLVFF (8), SEQ ID NO.: 7 LPFFD (30), SEQ ID NO.: 8 GVVIN, SEQ ID NO.:9 RVVIA (31) have been used to disrupt Aβ fibril formation andneurotoxicity. These pentapeptides have the same or similar residues assegments of Aβ essential for fibril formation, bind to Aβ, and alter thestructure that Aβ adopts (30). Generally, 1 to 1 molar ratios of peptideinhibitors to Aβ are needed in order to effectively prevent fibrilformation (8). Amphipathic molecules such ashexadecyl-N-methylpiperidinium (HMP) bromide or sulfonated moleculessuch as Congo Red have also been used to prevent Aβ fibril formation andtoxicity.

FIG. 8 is a nucleic acid sequence of an Hsp20 for use with the presentinvention (SEQ ID NO.: 1).

FIG. 9 is the amino acid sequence of an Hsp20 (SEQ ID NO.: 2) for usewith the present invention in accordance with SEQ ID NO.: 1.

FIG. 10 is a predicted secondary structure for Hsp20, which predictsthat Hsp20 is likely to contain a large amount of beta sheet secondarystructure. CD measurements were carried out to determine if thesestructures are present in the Hsp20 studies herein. Based on thepredicted structure, subfragments of hsp20 may be produced to determineif they may have the same activity as the whole protein. For example, ithas been determined that the Hsp20 active form is likely to be a dimer.As such, deletion scan may be conducted in which a small number ofresidues, e.g., beginning with the N-terminal 5-10 amino acid, todetermine if the Hsp20 is able to form dimers. Dimers may be detectedusing, e.g., physical observation (antibody capture assays, magneticbead binding, electron microscopy using gold beads of different sizes)or read out assays, e.g., fluorescence resonance electron transfer(FRET), surface plasmon resonance effects, yeast two hybrid, etc. Infact, some inactive or partially active forms of the monomer and/ordimer are also useful as controls and the like. For example, if thelarger inactive complexes are not allowed to form, the hsp20 will beactive over a much wider concentration range. Studies conducted to date(data not shown), for example, indicate that the intermediate forms ofamyloid are the likely target for binding of hsp20. It appears to be a10-mer in the first set of studies.

Circular Dichroism Measurements. FIG. 11 is a graph of CD measurementscarried out on an AVIV model 62. The wavelength scan was performed atvarious temperatures from 250 to 200 nm reading every 0.5 nm andaveraging each reading for 5 seconds. Circular Dichroism revealed a highbeta sheet secondary structure, typical of α-crystallin domains. In vivostudies have revealed properties unique to this α-crystallin.

As described hereinabove, the heat shock protein, Hsp20 from B. bovisprevents Aβ aggregation and toxicity at very low concentrations of smallheat shock protein relative to Aβ(1:1000). A series of studies wereconducted to examine the mechanism of Hsp20 activity and compare it toactivity of other small heat shock proteins. The aggregation andtoxicity prevention properties of two other small heat shock proteins,Hsp17.7 from carrot, and human recombinant Hsp27 were examined. BothsHsps inhibited Aβ1-40 aggregation but not toxicity. Electron microscopeimages of Hsp-Aβ complexes that formed under conditions and where bothaggregation and toxicity prevention were observed were compared toHsp-Aβ complexes that formed when only aggregation prevention was seen(data not shown). In cases where both aggregation and toxicityprevention were seen, a large ring structure was observed repeatedly,which was absent when aggregation was prevented, but toxicity was notprevented. For discussion, but by no means a limitation to thisinvention, it may be postulated the Hsp20-Aβ interaction leads to bothtoxicity and aggregation prevention. The present invention providesconstructs, vectors, proteins, methods and the like for preventingprotein aggregation and toxicity of Aβ as well as detailed instructionsfor designing the next generation of Aβ aggregation inhibitors to beused in AD.

β-Amyloid (Aβ)-(1-40) was purchased from AnaSpec (San Jose, Calif.) andBiosource International (Camarillo, Calif.). Recombinant human HeatShock Protein 27 (Hsp27) was purchased from MBL InternationalCorporation (Woburn, Mass.). Human neuroblastoma SH-SY5Y cells (ATCCnumber: CRL-2266) were purchased from ATCC (Manassas, Va.). Cell culturereagents were purchased from Invitrogen Life Technologies (Carlsbad,Calif.). Congo red was purchased from Fisher Chemicals. (Pittsburgh,Pa.). All other chemicals, unless otherwise specified Ire obtained fromSigma-Aldrich Co.

Heat Shock Protein 20 (Hsp20) Preparation Hsp20. was isolated fromBabesia bovis, [12] and Hsp20 with the N-terminal polyhistidine fusionprotein was produced in recombinant E. coli. Hsp20 was prepared bygrowing cells to OD₆₀₀ of 0.5 followed by induction with 1 mM IPTG for 5hours, and removal of media. The cells were then lysed in PBS using aFrench pressure cell and the insoluble His-Hsp20 pellet collected. Thepellet containing His-Hsp20 was suspended in Denaturing Binding Buffer(Invitrogen) containing 8M urea and incubated with Probond resin for 1hour to allow binding of His-Hsp20 to the nickel chromatography resin.The protein was eluted from the resin using 100 mM EDTA, dialyzedovernight into PBS pH 7.0, 20% glycerol and frozen at −80° C. Proteinpurity and molecular weight were confirmed by SDS PAGE.

Heat Shock Protein 17.7 (Hsp17.7) Preparation. The gene, encodingHsp17.7 from carrot, was cloned in E. coli (M. K. Malik, J. P. Slovin,C. H. Hwang, and J. L. Zimmerman, Modified expression of a carrot smallheat shock protein gene, hsp17.7, results in increased or decreasedthermotolerancedouble dagger, Plant J. 20 (1999) 89-99). E. coli wasgrown in the LB media (Tryptone (10 g/L), Yeast Extract (5 g/L) and NaCl(10 g/L)) with 50 μg/ml kanamycin broth on the wheel for aerationovernight. The tubes of LB broth were inoculated at a 1:200 dilution andput on wheel at 37° C. 100 mM IPTG was added to cells in order to induceHsp17.7 when E. coli reached an OD600 of 0.6. E. coli was put back onthe wheel and Hsp17.7 was expressed at 37° C. overnight. The cells wereharvested by centrifugation at 6,000 rpm for 10 minutes and frozen at−20° C. overnight. The cells were lysed with 20 mM imidazole and 10 minsonication. Hsp17.7 was purified by using a metal affinity column(POROS® MC) (Framingham, Mass.) with copper chromatography resin. (SelfPack™ POROS® 20MC Media) (Framingham, Mass.) The protein was eluted bypH gradient from pH 7.4 (Phosphate buffer, 50 mM NaH2PO₄, and 300 mMNaCl) to pH 4.5 (Phosphate buffer). Protein purity and molecular weightwere confirmed by size exclusion chromatography.

Protein Sample Preparation. Aβ1-40 was dissolved in 0.1% (v/v)Trifluoroacetic acid (TFA) solvent at the concentration of 10 mg/ml.This solution was incubated at room temperature for 20˜30 minutes inorder to completely dissolve the Aβ. Filtered phosphate-buffered saline(PBS, 4.3 mM Na₂HPO₄, 137 mM NaCl, 2.7 mM KCl and 1.4 mM KH₂PO₄, pH 7.4)was added to the Aβ solution to make the final concentrations used inexperiments. For cell viability assays, MEM medium was used instead ofPBS buffer. Aβ samples were mixed on the rotator at 18 rpm and 37° C.,and samples were taken out as a function of time. sHsps were added tothe Aβ samples before the samples were incubated (prior to aggregation).

Congo Red Binding. Congo red was dissolved in PBS at the concentrationof 120 μM and syringe filtered. The Congo red solution was mixed withprotein samples at 1:9 (v/v) ratios to make the final concentration ofCongo red 12 μM. After a short vortex, the mixtures were incubated atroom temperature for 3040 minutes. Absorbance measurements from 400 nmto 700 nm were taken (UV-Vis spectrometer model UV2101, Shimadzu Corp.;Kyoto, Japan). Alternatively, Congo red absorbance was read at 405 nmand 540 nm using an Emax Microplate Reader (Molecular Devices,Sunnyvale, Calif.). In both cases, PBS buffer was used as a blank. Theconcentration of Aβ fibrils was estimated from Congo red binding viaequation (1):

[Aβ _(FIB)]=(⁵⁴¹ A _(t)/4780)−(⁴⁰³ A _(t)/6830)−⁴⁰³ A _(CR)/8620)  (1)

where ⁵⁴¹A_(t) and ⁴⁰³A_(t) are the absorbances of the Congo red-Aβmixtures at 541 nm and 403 nm, respectively, and ⁴⁰³A_(CR) is theabsorbance of Congo red alone in phosphate buffer. [27] In Microplatereader, absorbances at 405 nm and 540 nm were assumed to be same asthose at 403 nm and 541 nm.

Aβ Turbidity Assay. Protein samples (100 μM Aβ and 100 μM Aβ+sHsps) wereprepared as described in Protein Sample Preparation. At every 30 minutesuntil 8 hours, turbidity of protein samples was monitored at 405 nmusing UV-Vis spectrometer (model UV2101, Shimadzu Corp.; Kyoto, Japan).PBS buffer solution was used as a blank.

Electron Micrograph (EM). 200 μl of Aβ peptide solution, prepared asdescribed above, was mixed, placed on glow discharged grids, and thennegatively stained with 1% aqueous ammonium molybdate (pH 7.0). Gridswere examined in a Zeiss 10 C transmission electron microscope at anaccelerating voltage of 80 kV. Calibration of magnification was donewith a 2,160 lines/mm crossed line grating replica (Electron MicroscopySciences, Fort Washington, Pa.).

Cell Culture. SH-SY5Y cells were grown in Minimum Essential Medium (MEM)supplemented with 10% (v/v) Fetal Bovine Serum (FBS), 25 mM Sodiumbicarbonate, 100 units/ml penicillin, and 100 mg/ml streptomycin. Cellswere cultured in a 5% (v/v) CO₂ environment at 37° C. incubator. Lowpassage number cells were used (<p20) in all experiments to reduceinstability of cell line.

Biological Activity Assay. For biological activity tests, SH-SY5Y cellsat a density of 1×10⁶ cells/mL1 were grown in 96 well plates. Cells werefully differentiated by addition of 20 ng/mL NGF for 8 days. Aβ samplesin MEM medium were added to the differentiated SH-SY5Y cells and thecells were incubated with Aβ samples at 37° C. for 2 hours. Negativecontrols (cells in medium with no Aβ) and positive controls (cellstreated with 800 μM H₂O₂ in 50% (v/v) medium for 2 hours) were alsoprepared. At least 3 wells were prepared for each Aβ treatment, and eachpositive and negative control.

Cell viability was determined by using the fluorescent nucleic acid dye,propidium iodode (PI). PI is mostly used fluorescent dye for stainingDNA in cells. PI can enter the dead cells and bind DNA in dead or lateapoptotic cells. PI is a DNA specific dye and can not cross the membraneof viable cells. In order to stain the dead cells, Aβ treated cells werewashed with PBS 1˜2 times and 150 μL of enzyme free dissociation bufferfrom Gibco (Carlsbad, Calif.) was added to the cells in order to detachSH-SY5Y cells from the surface. The cells were incubated at 37° C. forapproximately 5 minutes. 5 μL of 33 μM PI was added to the cells andcells were incubated for 15˜20 minutes at room temperature in the dark.Stained cells in 96 well plates were loaded in the FACS array (BDFACSArray, BD Bioscience; San Jose, Calif.) and fluorescence histogramsfor cells were obtained. To set up gates for the cell viability assays,2 sets of staining controls were prepared, unstained cells, and cellsstained with PI alone. Cells not stained with PI dye were taken as livecells, and the relative cell viabilities were calculated using equation(2).

$\begin{matrix}{{{Relative}\mspace{14mu} {Cell}\mspace{14mu} {Viability}\mspace{14mu} (\%)} = {\frac{\left( {L.C._{sample}{- {L.C._{H\; 2O\; 2}}}} \right)}{\left( {L.C._{NControl}{- {L.C._{H\; 2O\; 2}}}} \right)} \times 100}} & (2)\end{matrix}$

where L.C._(sample) is live cells (%) of cells treated with Aβ andsHsps, L.C._(Ncontrol) is live cells (%) of negative control, andL.C._(Aβ) is live cells (%) of cells treated with only Aβ.

Statistical Analysis. For each study, at least 3 independentdeterminations were made. Significance of results was determined via thestudent t test with p<0.05 unless otherwise indicated. Data are plottedas the mean plus or minus the standard error of the measurement.

Effect of Hsp20 on Aβ Aggregation and Toxicity. Aβ is toxic toneuron-type of cells when in aggregated form. Results to date indicatedthat His-Hsp20 has been very promising as an Aβ aggregation and toxicityinhibitor (S. Lee, K. Carson, A. Rice-Ficht, and T. Good, Hsp20, a novelalpha-crystallin, prevents A-beta fibril formation and toxicity, ProteinSci. 14 (2005) 593-601). Congo red binding was used as an indicator ofextended β-sheet structure and changes in Aβ aggregation in thisresearch. As shown in FIG. 12A, His-Hsp20 prevents Aβ aggregation,showing the characteristic U-shape pattern. At lower His-Hsp20concentrations than 0.1 μM, Aβ fibril formation decreases as a functionof His-Hsp20 concentration. However, His-Hsp20 becomes less effective atpreventing Aβ fibril formation in the higher concentrations. In FIG.12A, we also show the effect of His-Hsp20 on Aβ toxicity. His-Hsp20prevents Aβ toxicity over wide range of concentrations. SH-SY5Y cellstreated with the Hsp20-Aβ mixture shows higher cell viabilities(61.3%±2.6%-78.0%±3.6%) than those treated with 100 μM Aβ (45.3%±1.7%).Statistically, cell viabilities of Aβ itself are different from all cellviabilities of Hsp20-Aβ mixtures. (all p values between Aβ and Hsp20-Aβmixture are less than 0.05).

Effect of Hsp17.7 on Aβ Aggregation and Toxicity. As shown in FIG. 12B,Congo red result shows that His-Hsp17.7, a small heat shock proteinderived from carrot, prevents Aβ aggregation at all concentrationsbetween 1 nM and 20 μM, without the apparent loss of activity at evenhigher concentrations (up to 20 μM). As for toxicities in FIG. 12B,addition of 20 μM Aβ to SH-SY4Y cells results in about 30% toxicity.Hsp17.7-Aβ mixtures have almost same toxicities like Aβ alone.Statistically the toxicities between Hsp17.7-Ab mixture and Aβ alone arenot different. (all p values are greater than 0.05 between Aβ sample andother samples containing Hsp17.7). Thus, unlike Hsp20, Hsp17.7 preventsAβ aggregation without preventing Aβ toxicity.

Effect of Hsp27 on Aβ Aggregation and Toxicity. FIG. 12C shows thebiological activity of Aβ when incubated with Hsp27 with near the sameeffectiveness and at similar concentration ranges as Hsp17.7. UnlikeHsp20, Hsp27 doesn't lose its ability to prevent Aβ aggregation at highconcentrations (5 μM of Hsp27). In the toxicity experiments shown inFIG. 12C, the samples containing Hsp27 incubated with Aβ have the sameor more toxicity than the toxicity of Aβ alone. Statistically, cellviabilities of 100 μM Aβ with 5 nM−1 μM Hsp27 are not different fromthat of 100 μM Aβ. (all p values are greater than 0.05) 1 nM and 5 μMHsp27 increases the toxicity of 100 μM Aβ. In other words, Hsp27 doesn'tprotect the cells from the Aβ toxicity.

Kinetics of sHsps-Aβ Interaction (Congo red and Turbidity). For Hsp20 tobe a useful aggregation inhibitor (or model for an aggregationinhibitor) for Aβ, it is important to discern if Hsp20 simply slows therate of aggregation of Aβ, or actually alters the aggregation pathway ofAβ. To that end, kinetics of Aβ aggregation were measured as assessedvia Congo red binding and turbidity in the presence and absence ofsHsps. As seen in FIG. 13, Aβ aggregation increases slowly at first,followed by a period of rapid increase in fibril content, whicheventually saturates over time. When Hsp20 is added at the beginning ofaggregation, fibril formation and Congo red binding follow a verydifferent pattern. Initially, aggregation is accelerated, followed by arapid loss in fibril content and ability to bind Congo red. Theseresults suggest that Hsp20 does not simply slow the rate of Aβaggregation, but actually changes the fibril formation pathway, possiblysequestering a fibril initiating species that forms early duringaggregation and preventing further fibril growth. Hsp 17.7 and Hsp27also change the fibril formation pathway. However, both Hsp 17.7 andHsp27 don't show accelerated aggregation in the early incubation timelike Hsp20. Turbidity assay in FIG. 14 shows more drastic differencesbetween Hsp20 and other sHsps. Aβ fibril formation follows acharacteristic sigmoidal curve, with a lag phase at early incubationtimes, followed by a fibril growth phase, then a saturation phase. Aβinitially mixed with Hsp20 shows higher intensities at earlierincubation time between 0 and 4 hours, which means Hsp20-Aβ mixtureforms big species. In Congo red result, Hsp20-Aβ mixture at 2 hoursshows more Cong red binding than other samples. Both results suggestthat Hsp20-Aβ mixture forms big size species with β-pleated sheet atearlier incubation time. Hsp27-Aβ mixture shows higher intensity at 1hour, which doesn't make big differences after that. Hsp17.7-Aβ mixturedoesn't show higher intensity at earlier incubation time.

Ability of His-Hsp20 To Prevent Toxicity of Aβ with Time. In order toassess the ability of His-Hsp20 to prevent Aβ toxicity, His-Hsp20 wasadded to Aβ samples prior to Aβ aggregation, and toxicity of theHsp20-Aβ mixtures were tested as a function of time after incubation. Asseen in FIG. 14, His-Hsp20 prevents Aβ toxicity. Statistically, cellviabilities of Hsp20-Aβ mixture at 2, 4, and 8 hours are not differentfrom negative control (p=0.862, 0.087, and 0.140 for 2, 4, and 8 hoursrespectively, all p values are greater than 0.05), which means His-Hsp20protects the cells from Aβ toxicity completely. At 2, 4, and 8 hours, Aβshows only 50-70% cell viability compared to negative control. At 24hours, Hsp20-Aβ mixtures have cell viability of about 80%, whereas Aβshows only about 40% cell viability. Although His-Hsp20 doesn't preventAβ toxicity completely at 24 hours, His-Hsp20 still reduces the Aβtoxicity (viability is statistically different between Aβ and Hsp20-Aβmixture).

Electron Micrograph (EM) Images of Aβ with Small Heat Shock Proteins. Tofurther exam the relationship between Aβ structure, sHsp-Aβinteractions, and aggregation and toxicity prevention activities,electron microscopy was used to examine structures formed by Aβ in thepresence of the small heat shock proteins. In one set of studies,structures formed by 100 μM Aβ and 0.1 μM His-Hsp20 were examined as afunction of time during incubation with mixing, analogous to the kineticdata shown in FIG. 13. As early as 1-hour after incubation, Aβ and Hsp20begin to complex ring-like structures. (data not shown).

As seen in FIG. 15, a better defined multi-ring structure is observed attwo hours. The appearance of this large multi-ring structure correspondsto the maximum in Congo red binding seen in Hsp20-Aβ mixtures seen inFIG. 13. At later times, the ring structures seem to fall apart, withfew rings observed in micrographs at 24 hours after incubation. (datanot shown). The large multi-ring structure is only seen in micrographsof Aβ and Hsp20 when 100 μM Aβ and 0.1 μM Hsp20 are used. When the sameconcentration of Aβ, but higher and lower concentrations of Hsp20 areused (1 nM, 0.01 μM, 1 μM, and 5 μM Hsp20), the complex ring structureis not observed (data not shown). As seen in FIG. 16, similar electronmicroscopy studies were performed with Aβ and Hsp17.7. At no time duringincubation of Aβ and Hsp17.7 are the large multi-ring complexes observedin micrographs. Also examined were structures formed 24 hours afterincubation of Aβ and Hsp17.7, as these structures would represent thecomplexes added to cells in culture for toxicity assays. Some isolatedfibrils from Hsp17.7-Aβ mixture are observed at 24 hours (data notshown), which is very different from Hsp20-Aβ mixture at 24 hours.Differences in these structures might point to clues as to thedifferences in structures formed and their relationship to celltoxicity.

In Alzheimer's disease (AD) and in dementia associated with Parkinson'sdisease, an increased expression of Hsp27 and of αβB-crystallin wasfound (T. Iwaki, A. Kume-Iwaki, R. K. Liem, and J. E. Goldman, AlphaB-crystallin is expressed in non-lenticular tissues and accumulates inAlexander's disease brain, Cell 57 (1989) 71-78; K. Renkawek, G. J.Stege, and G. J. Bosman, Dementia, gliosis and expression of the smallheat shock proteins hsp27 and alpha B-crystallin in Parkinson's disease,Neuroreport 10 (1999) 2273-2276). These results suggest that chaperoneactivity of heat shock proteins may contribute to the pathogenesisassociated with protein aggregation in Alzheimer's disease (Y. C. Kudva,H. J. Hidding a, P. C. Butler, C. S. Mueske, and N. L. Eberhardt, Smallheat shock proteins inhibit in vitro A beta (1-42) amyloidogenesis, FEBSLett. 416 (1997) 117-121).

Small heat shock proteins (sHsps) are expressed in almost all organismswhen cells become stressed during exposure to unfavorable environments.Small heat shock proteins exist as large oligomeric complexes of 300-800kDa with monomeric molecular mass of 15-43 kDa (J. I. Clark, and P. J.Muchowski, Small heat-shock proteins and their potential role in humandisease, Curr. Opin. Struct. Biol. 10 (2000) 52-59; T. H. MacRae,Structure and function of small heat shock/alpha-crystallin proteins:established concepts and emerging ideas, Cell Mol. Life. Sci. 57 (2000)899-913; I. P. van den, D. G. Norman, and R. A. Quinlan, Molecularchaperones: small heat shock proteins in the limelight, Curr. Biol. 9(1999) R103-105.). The primary roles of sHsps are to stabilize otherproteins under stress conditions and protect them from aggregation (J.M. Bruey, C. Ducasse, P. Bonniaud, L. Ravagnan, S. A. Susin, C.Diaz-Latoud, S. Gurbuxani, A. P. Arrigo, G. Kroemer, E. Solary, and C.Gamido, Hsp27 negatively regulates cell death by interacting withcytochrome c, Nat. Cell. Biol. 2 (2000) 645-652). sHsps are composed ofthree parts in the primary sequence, the N-terminal domain, theα-crystallin domain, and the C-terminal extension. The α-crystallindomains, highly conserved 80-100 amino acid sequences located in theC-terminal regions, are very important for substrate binding. [34] Otherresearch groups showed that αB-crystallin prevented Aβ fibril formationin vitro, but not the toxicity (G. J. Stege, K. Renkawek, P. S.Overkamp, P. Verschuure, A. F. van Rijk, A. Reijnen-Aalbers, W. C.Boelens, G. J. Bosman, and W. W. de Jong, The molecular chaperonealphaB-crystallin enhances amyloid beta neurotoxicity, Biochem. Biophys.Res. Commun. 262 (1999) 152-156). Human Hsp27 inhibited fibril formationof Aβ1-42 in vitro, but it was less effective on the pre-formed amyloid.

In FIG. 12A, Hsp20 prevented Aβ aggregation over 80% in the Hsp20concentration of 1 nM-1 μM, and also reduced Aβ toxicity in this ranges.FIGS. 12B and 12C show that both Hsp 17.7 and Hsp27 prevent Aβaggregation but not toxicity. Therefore, it was found that Hsp17.7 andHsp27 prevents only Aβ1-40 aggregation, whereas Hsp20 prevents Aβ1-40aggregation and toxicity. The α-crystallin domains of these sHsps couldbe the crucial parts of the proteins in preventing Aβ aggregationbecause the α-crystallin domains are well-conserved regions and arefound in Hsp20, Hsp17.7, and Hsp27. The results of sequence homologiesusing the software CLUSTALW (EMBL-European Bioinfomatics Institude) showthat the sHsps lack sequence homology in both the N-terminus, andC-terminal extension. (32%—αB-crystallin and Hsp27, 15%—αB-crystallinand Hsp17.7, 8%—αB-crystallin and Hsp20, 17%—Hsp20 and Hsp17.7, 7%—Hsp20and Hsp27, and 7%—Hsp17.7 and Hsp27). These differences could result indifferent binding affinity between sHsp and Aβ or in differentmechanisms by which when they prevent Aβ aggregation. In FIG. 13 andFIG. 14, sHsps-Aβ mixtures don't change the Congo red and turbidity withtime in the saturation phase, which are parallel to Aβ. It suggests thatall sHsps prevent Aβ aggregation by changing Aβ aggregation pathwaysrather than slowing down Aβ aggregation. In FIG. 13 and FIG. 14, Hsp20shows very different pattern to prevent Aβ aggregation from other sHsps.One of the possible explanations is that Hsp20 has stronger bindingaffinity with Aβ than other sHsps. Different binding affinity results inHsp20 binding to Aβ in earlier stage of Aβ aggregation which are nottoxic species to the cells, whereas other sHsp bind to Aβ in later stageof Aβ aggregation. As a result, Hsp20 prevents Aβ aggregation more thanother sHsp in FIG. 12A, 12B and FIG. 12C.

Apparently, the large multi-ring formation of Hsp20 with Aβ seems to berelated to the ability of Hsp20 to prevent both Aβ aggregation andtoxicity. FIGS. 13, 14 and 15 indicate that only Hsp20 forms a largemulti-ring structure with Aβ (at 2 hours in mixing condition), whereasthe other sHsps were not observed to form ring structures. Thestoichiometric binding between Hsp20 and Aβ could be associated with thelarge multi-ring formation and optimum activity of Hsp20 in preventionof Aβ aggregation. In order to know the effect of Hsp20 assembly on Aβaggregation, a novel Hsp20 construct was prepared that was missing 11residues from the C-terminus that are partly responsible for Hsp20assembly. This form of Hsp20 forms stable dimers under most conditions.The activities of the non-aggregating form of Hsp20 and the aggregatingHsp20 were compared, and find little difference in their aggregationprevention and toxicity behaviors (data not shown). These resultssuggest that the binding ratio (stoichiometric binding) between Hsp20(dimer) and Aβ is more important in Aβ aggregation prevention than Hsp20assembly. Other sHsps could follow different mechanism via assembly fromHsp20. First, sHsps could assemble to form oligomeric complexes such ashollow cylindrical or spherical structure, and then sHsp assembliesprotect the hydrophobic parts of Aβ from aggregation.

These results demonstrate that Hsp20 may have a unique mechanism ofinteraction with Aβ that results in different activity in Aβ toxicityprevention relative to other sHsps.

FIG. 14 shows that Hsp20-Aβ mixture has the same cell viabilities asnegative control until 8 hours, although Hsp20 doesn't protect the cellscompletely from Aβ toxicity at 24 hours. Again, Hsp20 can delay Aβtoxicity via a unique mechanism of interaction with Aβ. In Alzheimer'sdisease, even delaying Aβ aggregation onset or slowing its progressionmight be therapeutically useful, as disease onset is late in life.

The kinetics of amyloid formation changes in the presence of hsp20. Itis not simply a slowing of the process but the use of a differentpathway.

FIG. 18 shows the dynamic relationship of the oligomeric states of hsp20using FPLC analysis of 29 μM hsp20 in 500 mM NaCl (13.69=16mer; 15.05=1Omer; 16.85=hexamer; 17.99=tetramer; 20.18=dimer; 22.72=monomerDimer=15% of total).

Kinetics of Hsp20 fibril prevention activity. Next, it was determinedwhether Hsp20 simply slowed the rate of aggregation of Aβ or if italtered the aggregation pathway such that non toxic species were formed.This was an important question, as if Hsp20 simply slowed the rate ofaggregation, then it would be a much less attractive aggregationinhibitor than if it actually changed the aggregation pathway away fromtoxic species. As can be seen from FIG. 17, in physiological buffers, Aβaggregates until approximately 40% of the peptide is incorporated intofibrils by 10 hours, after which time no further increase in Congo redbinding is observed. However, in the presence of Hsp20, maximum Congored binding occurs within 4 hours, after which a loss in Congo redbinding is observed. Similar trends were observed in toxicity studies asa function of time with analogously prepared samples. These data suggestthat Hsp20 does not simply slow down Aβ aggregation, but alters the Aβaggregation pathway. A turbidity assay also reveals the same results.

Hsp20 exists in multimeric forms. The existence of the forms isconcentration dependent. Only the lowest concentrations with smalleroligomeric forms are active in blocking Aβ fibril formation.

Self-assembly of hsp20 into multimeric complexes. Among α-crystallinsthat have been well studied, a variety of oligomeric states have beenobserved and correlated with active and inactive states of the proteins.An analysis of the dynamic relationship of the oligomeric states ofhsp20 and their relationship to α-crystallin activity was conducted. Oneapproach that has been successful with the His-tagged protein, and whichwas largely unsuccessful with the larger MBP fused version is analysisof hsp20 at several concentrations through FPLC. Superose 6 resin(Pharmacia) and size exclusion standards have been used to create astandard curve of elution volume versus standard molecular weight. Whenpurified recombinant his-Hsp20 is applied to the column at intermediateconcentrations under high salt conditions, (29 μM) a number of discreteoligomeric states are observed (FIG. 19). Elution volumes correspond tothe following multimeric species: 13.69=16mer; 15.05=10mer; 16.85=6mer;17.99=tetramer; 20.18=dimer; 22.72=monomer. The dimeric form of theprotein constitutes approximately 15% of the total hsp20 applied to thecolumn.

At intermediate concentrations (29 μM, FIG. 19) the dimeric speciesconstitutes approximately 42% of the protein eluted from the column.When this same protein solution is diluted further to a finalconcentration of 0.1 μM, and applied to size exclusion chromatography,the dimer is the predominant species, constituting more than 95% of theeluted protein (not shown), suggestions that an equilibrium existsbetween species, and that the dimeric form of the protein may beproduced selectively by designing experiments at low concentrations ofHsp20. Alternatively, the production of a dimeric species stable athigher concentrations would be advantageous.

In an effort to find the smallest active piece of hsp20 with respect toprevention of ADH denaturation and Aβ fibril formation, hsp20 wastruncated at C the and N terminus. All truncated forms prevent ADHdenaturation. C-terminal truncation and full length prevent Aβ fibrilformation. The other truncated versions (N-terminal and N and Cterminal) may also be tested in the fibril assay. The truncated formswere made to stabilize hsp20 in the form which is most active.

Stabilization of specific complexes; alteration of the steady statelevel of specific species. Based on the X-ray crystallographic analysisof other alpha crystallins the putative hsp20 domains governing assemblyof dimers, dodecamers and larger ordered alpha crystalline structureswere identified. Using recombinant DNA technology truncations ofspecific regions of the hsp20 recombinant protein were constructed andtested to show: (1) the equilibrium distribution of oligomer of thetruncated protein, (2) activity in an ADH denaturation assay of thetruncated protein and, β) amyloid fibril prevention by the alteredprotein.

FIG. 20 is a sequence alignment of M.j-sHSP and B. bovis HSP20: Nterminal and C terminal deletions with the C-terminal deletion ofHSP20=EMRRVQIDAKA (SEQ ID NO.: 5).

Based on the analysis of an alpha crystalline derived from athermophilic bacteria by Laksanalamai, et al (2003), thecarboxy-terminal 12 amino acids of hsp20 were removed (FIG. 20) in orderto reduce or eliminate the assembly of dimers into dodecamers, thustrapping hsp20 in dimer configuration. When the recombinant protein wasapplied to FPLC (Superose 6 resin), approximately 42% of the preparationmigrated as a dimeric species on the column (FIG. 21). These results arein contrast to the full length hsp20 which at equilibrium demonstratesonly 1-2% dimer at similar concentrations (Lee, et al., 2005). Thecarboxy-terminal truncated protein preparation, highly enriched fordimeric species was tested in an ADH assay (FIG. 22) and found to beequivalent in activity to the native protein in that it preventedthermal denaturation of ADH at identical molar ratios (1:2 and 2:1,hsp20:Aβ). When tested in an Aβ assay to prevent fibril formation, apreliminary study showed its activity to also be similar to that of thenative preparation (FIG. 21). Further constructs have been made thatlack the N terminal 50 amino acids and would be predicted to formdodecamers but lack the ability to form higher order complexes. A thirdprotein lacks both the C-terminal 12 amino acids as well as theN-terminal 50 amino acids. The truncated proteins, which have an intactalpha crystalline domain, are virtually identical in its ability toblock ADH denaturation (FIG. 22).

A honeycomb pattern of Aβ-hsp20 complex is detected in EM when everanti-fibril activity is present (see FIG. 17). After approximately 2hours of incubating Aβ peptide in the presence of hsp20, an increase inturbidity is seen. This turbidity has proved transient as it thendecreases over time. An EM of that 2 hour time point reveals aninteresting “honeycomb” pattern which the inventors believe is anAβ-hsp20 complex. It is not known at this time what the significance ofthis complex is, however, it may lead to an alternate interactionpathway for Aβ that does not lead to amyloid formation.

FIG. 23 is a graph that shows that fibril formation of Aβ peptide in thepresence of full length and truncated hsp20.

The present invention includes a novel small heat shock protein withdemonstrable Aβ fibril formation and toxicity prevention activity.Biophysical characterization was performed on the small heat shockprotein to determine that it is most likely active as a small oligomeror dimer, which is formed most readily at low concentrations or elevatedtemperatures. The small heat shock protein disclosed herein preventedboth fibril formation and toxicity at very low mole ratios of heat shockprotein to Aβ, suggesting that a low abundance Aβ aggregationintermediate interacts with the small heat shock protein. These resultsshow that the small heat shock protein alters the aggregation pathway asopposed to simply altering the rate of aggregation. In addition, stablecomplexes of Aβ and the small heat shock protein were isolated, whichmay be used for Aβ aggregation/toxicity prevention.

FIG. 24 shows the amino acid sequences for the full length, aminoterminal truncated, carboxy terminal truncated, both the amino andcarboxy terminal truncated, a his-tagged full length, a his-tagged aminoterminal truncated, a his-tagged carboxy terminal truncated, and ahis-tagged the amino and carboxy terminal truncated proteins that weredeveloped for and are part of the present invention.

Table 1. Coding sequence of B bigemina Hsp 20. The his-tagged is highlyrelated to the B. bovis Hsp20. The full-length his-tagged version showsthe same protection of ADH aggregation at 58 deg. C. (same method asthat used for B. bovis HSP20).

Extra amino acids in the His-HSP20 clone:

met-gly-gly-ser-his(6)-gly-met-ala-ser-met-thr-gly-gly-gln-gln-met-gly-arg-asp-leu-TYR-asp(4)-lys-asp-pro-thr-leu-glu-asn-leu-tyr-phe-gln-gly-aftercleavage, only gly remains

Entire Coding sequence (including purification tag and TEV proteasesite):

MGGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPTLENLYFQGMSCIMRCNNSEQEVVIDEQTGLPVKNHDYTEKPSVIYKPSTIVPQNTILEIPPPKELETPITFNPTVDTFFDADTNKIVVLMELPGFSHNDITVECGLGELIISGPRPKDELYEKFGNNLDIHIRERKVGYFYRRFKLPHNALDKSVAVSYSNGILDIRIECSQFSEMRRIQIDGKA

NA coding sequence of above protein sequence:

ATGGGGGGTTCTCATCATCATCATCATCATGGTATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGGGATCTGTACGACGATGACGATAAGGATCCAACCCTTGAAAATCTTTATTTTCAAGGTATGTCGTGCATTATGAGGTGCAACAACTCCGAACAGGAGGTTGTCATCGATGAGCAGACGGGACTCCCAGTGAAGAACCACGACTACACTGAGAAGCCCTCTGTGATTTACAAGCCGTCGACCATTGTTCCTCAAAACACCATCCTTGAGATCCCTCCTCCCAAGGAACTGGAAACCCCTATCACCTTCAACCCCACCGTCGACACCTTTTTCGATGCTGACACCAACAAGATCGTTGTTTTGATGGAACTGCCTGGATTCAGCCACAACGACATCACTGTGGAGTGCGGTTTGGGAGAACTCATCATCAGCGGCCCCCGCCCCAAGGACGAGCTTTACGAGAAGTTCGGTAACAACCTTGACATCCACATCCGTGAGCGCAAGGTTGGTTACTTCTACAGGCGCTTCAAGCTCCCGCACAACGCCTTGGACAAGTCTGTTGCTGTGTCTTACTCCAACGGTATCTTGGACATCAGGATTGAGTGCTCGCAGTTCTCCGAGATGCGCCGCATCCAGATCGACGGCAAGGCAT AA

In FIG. 26, the multimeric state of HSP20 (full-length and variousdeletions) is analyzed by crosslinking The cross-linked samples are thenvisualized via electrophoresis on a denaturing 12% polyacrylamide-SDSgel. Briefly, the reaction is carried out in cross-linking buffer (1 mMDTT, 100 nM NaCl, 0.2 mM EDTA, 0.05% v/v NP40, 10% v/v glycerol, 25 mMHEPES, pH 8.0) and the diluted purified protein samples are mixed at aratio of 1:1 with Cross-linking Buffer. Next, 3 μl of 0.1%glutaraldehyde are added to 27 μl of protein. At 5, 10 and 20 minutes,10 μl aliquots are withdrawn and combined with 10 μl of tris-glycinebuffer (to quench the cross-linker). For loading on SDS-PAGE, an equalvolume of 2×SDS loading dye is added and the samples are boiled for 5minutes and ran on 12% SDS PAGE.

In the figures, high-order structures are seen forming at the top of thegel within 5 minutes. The monomeric protein is depleted over timeindicating its role in multimer formation. These figures demonstratethat the full-length forms high-order oligomers. Also, the C-terminaldeletion (12 aa), N-terminal deletion (50 aa) and N—C-terminal doubledeletion (62 aa) seem to still form very high order oligomers despitethe fact that ⅓ of the 177-residue protein has been removed. Thisobservation along with the fact that the C-terminal deletion has noaffect on its amyloid protection activity or ADH aggregation protectionby the N—, C— and N—C-terminal deletions suggest that a much smallerregion of the protein is responsible for the activities observed(specifically aggregation and amyloid protection).

FIG. 25 is a gel that shows the effect of the truncations (as listed) onthe target protein, and that the monomeric protein is depleted over timeindicating its role in multimer formation. FIG. 26 is a gel that showsthe effect of the truncations (as listed) on the target protein, andthat the monomeric protein is depleted over time indicating its role inmultimer formation.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

-   1. Lambert, M. P., Barlow, A. K., Chromy, B. A., Edwards, C., Freed,    R., Liosatos, M., Morgan, T. E., Rozovsky, I., Trommer, B.,    Viola, K. L., Wals, P., Zhang, C., Finch, C. E., Krafft, G. A. &    Klein, W. L. (1998) Proc Natl Acad Sci USA 95, 6448-53.-   2. Iversen, L. L., Mortishire-Smith, R. J., Pollack, S. J. &    Shearman, M. S. (1995) Biochem J311 (Pt 1), 1-16.-   3. Wang, S. S., Becerrα-Arteaga, A. & Good, T. A. (2002) Biotechnol    Bioeng 80, 50-9.-   4. Ward, R. V., Jennings, K. H., Jepras, R., Neville, W., Owen, D.    E., Hawkins, J., Christie, G., Davis, J. B., George, A.,    Karran, E. H. & Howlett, D. R. (2000) Biochem J348 Pt 1, 137-44.-   5. Dahlgren, K. N., Manelli, A. M., Stine, W. B., Jr., Baker, L. K.,    Krafft, G. A. & LaDu, M. J. (2002) J Biol Chem 277, 32046-53.-   6. Cairo, C. W., Strzelec, A., Murphy, R. M. &    Kiessling, L. L. (2002) Biochemistry 41, 8620-9.-   7. Ghanta, J., Shen, C. L., Kiessling, L. L. & Murphy, R. M. (1996)    J Biol Chem 271, 29525-8.-   8. Pallitto, M. M., Ghanta, J., Heinzelman, P., Kiessling, L. L. &    Murphy, R. M. (1999) Biochemistry 38, 3570-8.-   9. Lansbury, P. T., Jr. (1997) Curr Opin Chem Biol 1, 260-7.-   10. Tjernberg, L. 0., Naslund, J., Lindqvist, F., Johansson, J.,    Karlstrom, A. R., Thyberg, J., Terenius, L. & Nordstedt, C. (1996) J    Biol Chem 271, 8545-8.-   11. Kudva, Y. C., Hidding a, H. J., Butler, P. C., Mueske, C. S. &    Eberhardt, N. L. (1997) FEBS Lett 416, 117-21.-   12. Stege, G. J., Renkawek, K., Overkamp, P. S., Verschuure, P., van    Rijk, A. F., Reijnen-Aalbers, A., Boelens, W. C., Bosman, G. J. & de    Jong, W. W. (1999) Biochem Biophys Res Commun 262, 152-6.-   13. Brown, W. C., Ruef, B. J., Norimine, J., Kegerreis, K. A.,    Suarez, C. E., Conley, P. G., Stich, R. W., Carson, K. H. &    Rice-Ficht, A. C. (2001) Mol Biochem Parasitol 118, 97-109.-   14. Klunk, W. E., Jacob, R. F. & Mason, R. P. (1999) Anal Biochem    266, 66-76.-   15. Horwitz, J. (1992) Proc Natl Acad Sci USA 89, 10449-53.-   16. van den, I. P., Norman, D. G. & Quinlan, R. A. (1999) Curr Biol    9, R103-5.-   17. Bruey, J. M., Ducasse, C., Bonniaud, P., Ravagnan, L., Susin, S.    A., Diaz-Latoud, C., Gurbuxani, S., Arrigo, A. P., Kroemer, G.,    Solary, E. & Gamido, C. (2000) Nat Cell Biol 2, 645-52.-   18. Jakob, U., Gaestel, M., Engel, K. & Buchner, J. (1993) J Biol    Chem 268, 1517-20.-   19. MacRae, T. H. (2000) Cell Mol Life Sci 57, 899-913.-   20. van Montfort, R. L., Basha, E., Friedrich, K. L., Slingsby, C. &    Vierling, E. (2001) Nat Struct Biol 8, 1025-30.-   21. Horwitz, J. (2000) Semin Cell Dev Biol 11, 53-60.-   22. Hughes, S. R., Khorkova, 0., Goyal, S., Knaeblein, J., Heroux,    J., Riedel, N. G. & Sahasrabudhe, S. (1998) Proc Natl Acad Sci USA    95, 3275-80.-   23. Hatters, D. M., Lindner, R. A., Carver, J. A. &    Howlett, G. J. (2001) J Biol Chem 276, 33755-61.-   24. Abgar, S., Vanhoudt, J., Aerts, T. & Clauwaert, J. (2001)    Biophys J80, 1986-95.-   25. Lomakin, A., Teplow, D. B., Kirschner, D. A. &    Benedek, G. B. (1997) Proc Natl Acad Sci USA 94, 7942-7.-   26. Pallitto, M. M. & Murphy, R. M. (2001) Biophys J81, 1805-22.-   27. Yong, W., Lomakin, A., Kirkitadze, M. D., Teplow, D. B.,    Chen, S. H. & Benedek, G. B. (2002) Proc Natl Acad Sci USA 99,    150-4.-   28. McHaourab, H. S., Dodson, E. K. & Koteiche, H. A. (2002) J Biol    Chem 277, 40557-66.-   29. Liang, J. J. (2000) FEBS Lett 484, 98-101.-   30. Soto, C., Sigurdsson, E. M., Morelli, L., Kumar, R. A.,    Castano, E. M. & Frangione, B. (1998) Nat Med 4, 822-6.-   31. Hetenyi, C., Szabo, Z., Klement, E., Datki, Z., Kortvelyesi, T.,    Zarandi, M. & Penke, B. (2002) Biochem Biophys Res Commun 292,    931-6.

What is claimed is:
 1. An isolated and purified polypeptide comprising aportion of an α-crystallin polypeptide comprising one or more β-pleatedsheets that inhibits neurotoxicity and amyloidogenesis.
 2. Thepolypeptide of claim 1, wherein the polypeptide is non-human.
 3. Thepolypeptide of claim 1, wherein the polypeptide is from a bovineerythrocyte parasite.
 4. The polypeptide of claim 1, wherein thepolypeptide is from a Babesia sp. bovine erythrocyte parasite.
 5. Thepolypeptide of claim 1, wherein the polypeptide is from a Babesia bovis.6. The polypeptide of claim 1, wherein the polypeptide comprises Hsp20.7. The polypeptide of claim 1, wherein the polypeptide comprises SEQ IDNO.:
 2. 8. The polypeptide of claim 1, wherein the polypeptide furthercomprises a His-tag.
 9. A method of making an α-crystallin polypeptidecomprising the steps of: culturing a host cell comprising a nucleic acidsequence encoding at least a portion of the polypeptide of SEQ ID NO.: 2under conditions wherein the host cell expresses the polypeptide andwherein the polypeptide exhibits α-crystallin activity, attenuatedneurotoxicity and inhibits amyloidogenesis.
 10. An isolated and purifiedheat shock protein comprising one or more β-pleated sheets thatmodulates neurotoxicity over about a one-thousand-fold concentrationrange by stabilizing an amyloidogenic protein.
 11. The protein of claim10, wherein the concentration range is between about 1 nM to about 5 μM.12. The protein of claim 10, wherein the molar ratios of heat shockprotein to amyloidogenic protein is between about 0.0005 to about 0.001.13. An isolated and purified portion of a polypeptide having asignificant similarity to the α-crystallin family of proteins and one ormore β-pleated sheets isolated from Babesia sp.
 14. An isolated orpurified heat shock protein truncated at the amino, carboxy or amino andcarboxy terminus Hsp20 protein that inhibits Aβ₁₋₄₀ fibril formation.15. A pharmaceutical composition for treating amyloidogenesiscomprising: an effective amount of a purified polypeptide withα-crystallin activity and one or more β-pleated sheets that inhibitsneurotoxicity and amyloidogenesis in pharmaceutically acceptablecarrier.
 16. An isolated and purified polypeptide comprising anα-crystallin, small, heat shock protein Hsp20 having one or moreβ-pleated sheets that inhibits neurotoxicity and amyloidogenesis. 17.The polypeptide of claim 16, wherein the polypeptide is from a Babesiabovis.
 18. The polypeptide of claim 16, wherein the polypeptide furthercomprises a His-tag.
 19. The polypeptide of claim 16, wherein thepolypeptide interferes with intermediate forms of amyloid.